Since the divalent cation, calcium, is involved in the regulation of excitation-contraction coupling in the cardiac muscle, alterations in the calcium-related activities in the cardiac sarcolemma will also bring abnormalities in the cardiac muscle. Thus the calcium binding to the cardiac sarcolemma has been measured. Calcium binding to the sarcolemma was measured by ultracentrifugation technique. Two classes of binding sites with dissociation constants of 2.0 times 10 minus to the 5th power M and 1.2 times 10 minus to the 3rd power M were identified. The capacities of the high and low affinity sites were 15 and 452 nmoles/mg respectively. These sites were not affected by treatment neuraminidase. The effects of various cations and drugs on calcium binding were studied. All cations tested inhibited calcium binding with the order of potency: trivalent greater than divalent greater than monovalent cations. Caffeine (10 to the minus 3rd power M) and ouabain (10 to the minus 8th power M) increased the capacity of the low affinity sites to 1352 and 837 nmoles/mg respectively. Verapamil (10 to the minus 7th power M), acidosis (pH 6.4), Mn ions (10 to the minus 5th power M) and ouabain (10 to the minus 4th power M) depressed the capacity of the low affinity sites to a range of 154-291 nmoles/mg. The dissociation constant of the high and low affinity sites and the capacity of the high affinity sites were not affected by the inotropic agents.